Chapter 6 Micropropagation of Date Palm Using Inflorescence Explants A.A. Abul-Soad Abstract Inflorescence-based micropropagation holds great potential for the multiplication of recalcitrant male and female date palm individual trees and cultivars of commercial interests with limited populations. This can be accomplished in a short time with minimal effort as compared to the traditional practice of using shoot-tip explants. The aim of this technique is to pave the way to use inflorescence explants to micropropagate date palm by direct formation of organs (somatic embryos or shoots) and avoiding most of the constraints that face the shoot tip like high percentage of contamination in the establishment stage, heavy browning, a long time for first cluster initiation, using a considerable number of offshoots and inability to micropropagate the elite palms in case no offshoots are available. The way to excise the immature inflorescence without damage to the mother tree, composition of the nutrient medium for direct organ initiation have remained hindrances to this technique over recent decades, in addition to the technicalities for proper handling of cultures inside the laboratory and successful shifting of plants to the greenhouse. We report for the first time an innovative method used to excise the immature inflorescence at a suitable stage for successful culture initiation. Spikelet explants are induced to produce shining globular structures without a callus phase. Also, explants were exceptionally able to develop direct shoots. Two types of organs developed on the differentiation medium, green shoots and intact somatic embryos. Only green shoots and multiple somatic embryos were subjected to proliferation at the multiplication stage. Well-rooted plantlets were hardened and successfully established in soil. Keywords฀ Acclimatization฀ •฀ In vitro฀ •฀ Inlorescence฀ •฀ Phoenix dactylifera L. • Regeneration A.A. Abul-Soad (*) Horticulture Research Institute, Agricultural Research Center, 9 Cairo Univ. St., Orman, Cairo, Egypt e-mail: adelaboelsoaud@gmail.com S.M. Jain et al. (eds.), Date Palm Biotechnology, DOI 10.1007/978-94-007-1318-5_6, © Springer Science+Business Media B.V. 2011 91 92 6.1 A.A. Abul-Soad Introduction The date palm (Phoenix dactylifera L.) is a dioecious, perennial monocotyledonous plant species belonging to the family Arecaceae. It is one of the oldest fruit tree crops and is primarily cultivated in North African and Middle Eastern countries. Flowers are borne on stalks growing among the leaves. The date palm has compound, branched flower stalks (inflorescences) bearing small whitish or creamy flowers (Fig. 6.1a). Traditionally, shoot tip explants from offshoots are used for various micropropagation protocols for both research and commercial production Fig. 6.1 Establishment of the inflorescence cultures. (a) Schematic illustration of 15 cm spathe components (1: Inflorescence base, 2: Spikelet base, 3: Spikelets, 4: Protective sheath), (b) The mother tree of excision (left), and subsequent season carrying regular bunches (right), note the number of bunches, (c) Initial spikelet explant, (d) Scanning Electron Microscopic of one floret 25× 6 Micropropagation of Date Palm Using Inflorescence Explants 93 purposes (Abul-Soad et al. 2002a,b; El-Hadrami and Baaziz 1995; Tisserat 1984b). The major disadvantage of this practice is scarification of the entire offshoot. Ultimately, this will hinder the micropropagation of superior individuals without offshoots or of commercially viable cultivars with limited numbers of available individual specimens. Inflorescence explants have proven promising and afford a needed alternative explant source for micropropagation of elite cultivars and rare individuals of date palm. We describe here a stepwise micropropagation procedure using inflorescence explants and compare their benefits with the use of shoot-tip explants; innovative ways to safely excise an immature inflorescence from an adult tree at the appropriate time; establishment of the initial explants on a starting medium after surface sterilization of the spathe; other details on rooting, hardening and field performance in fruiting. 6.2 Morphogenic In Vitro Responses of Date Palm Inflorescence Inflorescences of several palm species have been cultured in vitro. Since 1973, several researchers have attempted to culture palm inflorescences. Explants of female and male oil palm inflorescences were grown on culture media and usually plants regenerated normally without callus formation (Smith and Thomas 1973), whereas in date palm, a high auxin level was required (Eeuwens and Blake 1977). The addition of auxins in the culture media increases the frequency of visible expanded carpels developing from supposedly male date palm (Tisserat and DeMason 1980). Vestigial female date carpels on surviving male flowers become enlarged and quite prominent (Tisserat 1979). White friable callus usually was initiated from the floral bud strand (Tisserat et al. 1979). In some cases, roots and embryoids were initiated from explants of coconut inflorescence rachillae (Eeuwens 1978) and date palm (Tisserat 1979). Date palm ovules, carpel tissue, parthenogenetic endosperm and fruit stalks turned black within 24 h after culturing on nutrient media and subsequently died. Cultures of date palm floral bud reproductive tissue, especially male anthers, usually turned brown and died after a few weeks in culture (Reuveni et al. 1972). The tissue of choice in palms for obtaining embryogenic callus is either the actively growing lateral bud (when available) or the shoot tip (Tisserat 1981). Older tissues and organs do not respond well in culture, and callus produced from them usually has limited morphogenetic potential; e.g., mature leaf and flower bud callus produce only roots (Abul-Soad 2003; Tisserat 1984a). Tissue culture studies of date palm inflorescences have demonstrated the varied potential of female floral initials. Firstly, these initials were able to change from the floral state to vegetative outgrowths by different methods, all of which were correlated to the stage of differentiation at the time of excision. Secondly, they changed according to the composition of the initial nutrient medium, especially to the plant growth regulator formula used. Also, the sequence of nutrient media used is decisive 94 A.A. Abul-Soad (Abul-Soad 2003). These floral initials were able to develop directly into complete plantlets. Floral initials also had the potential for induction of adventitious buds or somatic embryos, which in turn developed into complete and separated plantlets (Drira and Al-Sha’ary 1993). Therefore, Abahmane et al. (1999) suggested that a micropropagation technique using floral tissues of floral spikes at an early stage of growth on culture media aided in converting these tissues from floral to vegetative. Moreover, this early stage can vary by cultivar, climatic conditions and nutritional status of the mother tree (Abul-Soad 2007a). On the contrary, Kriaa et al. 2007 envisaged a protocol to be based on the use of mature female flowers, taken at the latest developmental stage, before opening of the spathe. This strategy has an advantage of avoiding damage to the mother plant. The types of formations of well-responding inflorescence explants are of three types: (a) direct green shoots from stressed or long-term incubated initial explants (Abul-Soad et al. 2011), (b) direct embryogenic callus which will differentiate after maturation into direct somatic embryos or direct shoots (Abul-Soad et al. 2004a; Abul-Soad 2007a, 2009; Kriaa et al. 2007) and (c) unfriable callus (Abul-Soad et al. 2005; Feki and Drira 2007). In another trial carried out on male inflorescences, although the callus formed, it failed to form somatic embryos. This is perhaps related to the age of the explant source (Al-Khayri et al. 2007). In addition, cultured female flower initials developed into hermaphrodite flowers having stamens and anthers filled with pollen, besides the normal three carpels (Drira and Benbadis 1985) or green flowers without pollen (Abul-Soad 2003). In that way, morphologically-typical hermaphrodite flowers were obtained (Masmoudi et al. 2007). Thus far no laboratory has succeeded in commercially propagating date palms (male or female) from an inflorescence except in Al Ain City, UAE where male date palm has been commercially propagated (El-Korchi 2007). Also, female inflorescence was successfully used for commercial micropropagation of 12 date palm varieties in Date Palm Research Institute, Shah Abdul Latif Univ., Pakistan (Abul-Soad and Mahdi 2010). Most of the trials have faced some unknown hindrances at the experimental level. Some of these hindrances are the reproducible protocol by which huge numbers can be produced in a short time. The procedure which can be used for the inflorescence excision without any damage or dangerous for the mother tree at an early stage is sensitive. Producing plantlets through the callus phase can produce somaclonal variation, rather than the lengthy procedure and growth vigor of produced plants from callus. High auxin concentration can be used to induce the callus from inflorescence explants in the case of using mature or late age explants. An appropriate formula is not known which can induce the direct organs, either somatic embryos or green shoots, within a few months for the overwhelming majority of cultured explants. However, the manner of applying this technique is of paramount importance. It is conceivable that more than 100 cultivars can be managed and initially established within 1–2 months in one laboratory, with no wasted offshoots and significantly less efforts. On a commercial level, the Biotechnology Laboratory of Date Palm Research Institute, Khairpur, Pakistan succeeded in producing seven commercial Pakistani 6 Micropropagation of Date Palm Using Inflorescence Explants 95 cultivars in the first run. These cvs. were Dhakki, Gulistan, Aseel, Kurh, Kashowari, Dedhi and Gajar, most of which were healthy and of commercial interest to the date industry of Pakistan (Abul-Soad 2007b; Markhand and Abul-Soad 2010). One or two immature inflorescences were excised from targeted cultivars of the major date-growing areas in Pakistan (Khairpur and Dera Ismail Khan). These cultivars are represented by the early cvs. (Gajar and Kashowari), the major export cv. (Aseel), a cultivar with high price and international standards cv. (Dhakki) and low population cv. (Gulistan). For example, cv. Gulistan at Dera Ismail Khan was multiplied by traditional methods late in the last century, amounting to a few thousands and there is growing demand for its offshoots which cannot be met by the traditional offshoot-detachment method. Tens of thousands of plantlets were micropropagated within 2 years from only two small inflorescences (Abul-Soad and Mahdi 2010). 6.3 Benefits of Using Inflorescence Explants These benefits can be divided into two groups based on considerations related to in vitro and in vivo growth behavior. 6.3.1 Considerations Related to In Vitro Tissue Culture •฀ Fungal and bacterial contamination is very low. Despite surface sterilization with chemical substances, 90% of shoot-tip explants can be lost through contamination, particularly from endogenous bacteria. In contrast, inflorescence explants which are contaminant-free can reach up to 100% success (Abul-Soad 2007a; Abul-Soad et al. 2008). •฀ Minimal browning occurs. Wounding an explant in the shoot-tip technique causes secretion of phenolic compounds into the culture medium and prevents the growth of the initial explant and ultimately leads to death. Nevertheless it can be controlled by incubation in complete darkness and activated charcoal in the media especially during the initial stage. But the whole organ (spikelet) is typically used in this technique. •฀ Juvenility of inflorescence explants used which have high potential to differentiate quickly into organs. At the initial stages of growth, the explant has masses of meristematic tissue making it easy to express morphogenetic responses (Abul-Soad et al. 2011). •฀ A time-efficient protocol in which the shorter starting stage is only 2–6 months as compared to at least 6–18 months for organogenesis from shoot tip explants (direct pathway). •฀ Consequently, inflorescence explant cultures reduce the time scale of the entire production to 1–2 years as compared to 2–3 years with shoot tips. Also, the formation of organs with a minimal intervening callus phase avoids the risk of somaclonal variation. 96 6.3.2 A.A. Abul-Soad Considerations Related to In Vivo Open-Field Characters •฀ Micropropagation of recalcitrant cultivars, especially those existing in limited numbers (Kriaa et al. 2007) or rare individual female lines (Abdallah 2002), facilitate large scale production of plants for commercial uses; including some elite cultivars without any or with few offshoots. No method is known to reproduce such cultivars and lines except through inflorescence explants. •฀ Micropropagation of highly-selected and extremely-useful male lines (Al-Ghamdi et al. 2007) can be achieved. Fruit quality properties such as size, color and sugar content are dependant mainly on the pollen grain source (the metaxenia phenomenon). This means that not all heterozygous males are equal in their impact on fruit quality. There have been many studies and evaluation programs to select ideal male trees. These studies are without potential if there are no offshoots on the tree, i.e. no source for further propagation. The inflorescence technique is the only way currently available to reproduce such vigorous males (El-Korchi 2007). •฀ Micropropagation of cultivars resistant to epidemic diseases such as bayoud (Fusarium oxysporum f. sp. albdinis) and pests like the red palm weevil (Rhynchophorus ferrugineus F.). There are a few cultivars and lines exhibiting natural resistance to these maladies, but the trees typically produce no offshoots. It is reported that well-acclimatized plants derived from inflorescences of INRA-J19 and INRA-A6 clones were tested against bayoud disease (Abahmane 2007). 6.4 Excision of Immature Inflorescence Date palm is an important crop plant in arid and semiarid areas with a hot dry summer season and little rain in winter. Due to these conditions, date palm flowering occurs each year at the beginning of spring (Northern Hemisphere) and facilitates availability of immature female inflorescence (spathe) for tissue culture. The growth and development of small floral buds to reach the usual mature size of the female spathe is rapid. Their appearance on the tree is synchronized with increasing air temperatures in the early spring season. Typically, the flowering season takes 2–6 weeks for complete emergence of all spathes. The appropriate excision time for each cultivar should be investigated to determine a precise time for excision of spathe within that timeframe. The excision time is equally crucial to avoid sexual organ formation which occurs at the late stage of development. In view of the fact that carpels are the last phase of flower differentiation, an early stage of growth is required to avoid any sexual cells interrupting the organogenesis from somatic cells alone. The age of the explant plays an important role in this protocol. Some reports suggest using mature female flowers at the latest developmental stage of growth, just before the opening of the protective sheath (Kriaa et al. 2007). Others refer to using the inflorescence as soon as it emerges from among the leaves and the brown tip of the spathe becomes visible (Abahmane 2007). In a trial with a male tree from 6 Micropropagation of Date Palm Using Inflorescence Explants 97 Al-Ain City, the youngest possible spathes, 51–54 cm long, were excised, (El-Korchi 2007). Also, they referred to the source of inflorescences, offshoots of cv. Barhee approximately 1–1.2 m high; the inflorescence if present in the axil of the offshoot, was removed with its protective sheath (spathe) intact and refrigerated at 4˚C until used (Bhaskaran and Smith 1992). By contrast, the current innovative protocol recommends excision of the spathe while it is still hidden among the leaves (15 cm long). A method developed based on empirical experience was able to estimate the location of young spathes on the tree with a success rate 80–90%. The flower buds that will develop into inflorescences begin to emerge in reciprocal positions around the head of the tree, mainly above 4–5 older frond whorls. Thereafter, spathes emerge among the next 3–4 frond whorls (Abul-Soad 2003). Based on a preliminary study, one frond is selected and the adjacent 4–5 fronds peeled away. In the case of not finding the spathe, the operation should be aborted; otherwise this can cause the crown of the tree to fall off. Only a skilled person can induce the same tree to provide a maximum of 1–2 spathes. In another proven method, pruning of the outer mature fronds is carried out until reaching the first outer spathe. By cutting 36–40 mature fronds the loss of a paramount source of spathes occurs in the current season. Therefore, bunch thinning should be done to reduce the number of fruiting bunches from 15 to 20 down to only 4 bunches. The mother tree entirely recovered in the following season and the location of excision had almost disappeared (Fig. 6.1b). In both methods, the time of excision, cultivar and climatic conditions, particularly temperature, are variable factors that control the appropriate excision time, i.e. age of the spathe. The mother tree from which the spathe was excised lost only one bunch in that same year and entirely recovered the next year. Care must be taken to treat the wound with fungicide and pesticide to prevent any further infection or infestation from diseases or pests, in particular the red palm weevil. The inflorescence was excised from the mother plant in early spring, placed in a clean plastic cover and handled carefully from the open field to the laboratory. In the event of considerable distance between the laboratory and the location of the source material (spathes), the spathe can be conserved for 1–2 days in an ice chest. This step had no adverse impact on the survivability of the spathes. 6.5 Surface Sterilization of Spathes One of the worthwhile benefits of the inflorescence technique is the simple sterilization protocol to decontaminate explants. As soon as the entire inflorescence (spathe) has been brought into the laboratory, immediate surface sterilization should be carried out according to the following steps: •฀ Dipping into fungicide solution for 30 s, without any shaking. The most commonly used fungicide is Tobsin or Bennlit (systematic fungicide) solution 2 g l–1. •฀ Handle carefully spathe while washing under running tap water for 30–60 s. The water stream should be directed at the basal part of the spathe to remove dirt. 98 A.A. Abul-Soad •฀ Using 30% sodium hypochlorite (NaOCl) solution (16%) for 1–2 min. •฀ Washing with sterilized distilled water for 30–60 s one time without shaking. Cracks or cuts in the outer protective sheath of the spathe due to mishandling during excision, transfer to the laboratory or surface sterilization can increase the contamination rate to as much as 100%. Maximum care must be given to handling the spathe through all stages. Keeping the spathe undamaged results in definitely highly survival and of explants free of contaminants (Abul-Soad 2003, 2007a, 2009). This protocol avoids any direct contact between the surfactants and the spikelet explants. That is why the explants respond well to the nutrient medium. Most of other protocols (El-Korchi 2007) call for surface sterilization of the spikelet explants but not for the entire spathe (Abul-Soad 2007a; Abul-Soad and Mahdi 2010). 6.6 Establishment of Initial Cultures In a schematic illustration (Fig. 6.1a), the spathe can be divided histologically into four sections: inflorescence base, spikelet base, spikelet and protective sheath. The spikelet was the only explant component which responded well to different nutrient media (Fig. 6.1c). Other sections failed to produce even callus onto the callus induction medium (Abul-Soad 2003). The procedure of surface sterilization of the intact spathe involves the following steps: •฀ The outer protective sheath surface is sterilized and the spathe cut longitudinally from the middle like a T from only one side. The cut may be made in the central swollen portion of the spathe due to its softness. •฀ Spikelets cut, 3–4 cm long, from their base can be cultured. Spikelets are cut 2–3 cm long and placed horizontally on the sloping surface of the nutrient medium. Each of these should have 2-4 immature florets (Fig. 6.1d). •฀ All cultured tubes are incubated in a controlled growth room at 25 ± 2ºC in the dark. Incubated explants are subcultured every 3–4 weeks on the same starting culture medium, as described in Table 6.1. •฀ Well-responding explants are transferred to a maturation medium for 1-2 subcultures. •฀ Matured globular structures which are ready to differentiate and early-differentiated explants growing in the darkness are shifted onto the differentiation medium for 16 h photoperiod and under 2,000–3,000 lm m–2 conditions for 1-2 subcultures each for 4–6 weeks. Subsequently differentiated cultures are shifted to the multiplication stage or may be rooted directly. The age of a spikelet explant does not necessarily determine its length. The length of cultivar spikelets is variable within the same inflorescence or even among different inflorescences on a tree. Moreover, the spikelets are located within the spathe in a pyramid-like shape. The longest spikes are found in the center and are surrounded by shorter ones on the periphery. It seems that the endogenous hormones of the explant control the number of responding florets on the explant. The most suitable culture vessel is a test tube 6 Auxins 0.1 2.4-D + 0.1 IAA + 5.0 NAA 5.0 2,4-D 0.1 NAA 0.1 NAA 0.1 NAA Cytokinins – 1.0 2iP 0.1 Kinetin 0.05 BA Micropropagation of Date Palm Using Inflorescence Explants Table 6.1 Nutrient media composition used for inflorescence protocol and its sequence Composition (mg l–1) Medium Salts Additives 30000 Suc.c + 2200 Agar + 1400 Gel + Vit.d of 1. Starting Macro of B5a + Micro of MSb MS + 170 KH2PO4 + 100 Glutamine + 40 Ad.e 2. Maturation Macro of B5 + Micro of MS 30000 Suc. + 2200 Agar + 1400 Gel + Vit. of MS + 170 KH2PO4 + 100 Glutamine + 40 Ad. + 1500.0 ACf 3. Differentiation MS 30000 Suc. + 2200 Agar + 1400 Gel + Vit.of MS + 4. Proliferation MS 30000 Suc.+ 2200 Agar + 1400 Gel + Vit. of MS + 5. Rooting ¾ MS 50000 Suc. + 2200 Agar + 1400 Gel + 0.1 Ca-panthothianate + Vit. of MS + with 3000.0 AC. a B5: Gamborg et al. (1968) Nutrient Medium b MS Murashige and Skoog Medium (1962) c Suc. Sucrose d Vit. Vitamins e Ad. Adenine Sulfate f AC Activated Charcoal 99 100 A.A. Abul-Soad Fig. 6.2 Inflorescence morphogenesis. (a) Pro-embryogenic masses (PEMs) induction on the spikelet explant of cv. Dedhi after 3 weeks in culture onto modified MS basal medium supplemented with 1.0 mg l–1 NAA + 0.1 mg l–1 IAA + 0.1 mg l–1 2,4-D, (b) Shining globular creamy structures after 2–3 months in culture, (c) Maturation on activated charcoal medium in darkness, (d) Caulogenesis during differentiation process (25 × 150 mm). However, there was no significant difference according to the type of culture vessel, especially at the starting stage of culture initiation. Mostly there was one explant per tube; however, more than one explant can be cultured in a single tube but that may result in poor growth of cultures. Moreover, only rarely did one explant have all immature florets on it responding. One type of positive response is the formation of a globular shining structure, instead of the immature floret or cleaved from the floret. The color typically is creamy and sometimes bright white (Fig. 6.2a). It is worth mentioning that the sequence and manipulation of cultures is very important. This is related to the proper direction of each morphogenetic type to its subsequent suitable stage of growth and nutrient medium. For example, if the globular structures shift directly to a differentiation medium without maturation, most of them will not differentiate; nevertheless, there is no significant change in appearance before and after transfer to the maturation medium. As well as, transfer of the differentiated cultures to the multiplication or rooting stage directly according 6 Micropropagation of Date Palm Using Inflorescence Explants 101 to its shape to maximize the production. Individual cultures will go directly to the rooting stage to reduce the production time and give a whole plant, but multiplied cultures will go on to the multiplication stage to proceed with normal growth and produce more cultures. The time schedule of organogenesis from well-established initial explants was as follows: •฀ Shining globular creamy structure formation was at about after 2 months of culture, through 1–2 subcultures (Fig. 6.2b). •฀ Maturation of initial structures after 2–3 months, through 2–3 subcultures (Fig. 6.2c). •฀ Differentiation process at 1–2 months, through 1–2 subcultures (Fig. 6.2d). •฀ Proliferation phase of individual shoots and multiple somatic embryos. •฀ Multiplication and rooting stages. This can be extended from a few months to 1–2 years depending on simultaneously required plantlet numbers and their health. The first and second steps must be done in complete darkness for induction and maturation processes of organs, while other further steps can be done under illuminated conditions. It was observed that the presence of light during the initial phase disrupted the growth and increased the degree of browning. In the case of matured cultures failing to develop into organs, they must be returned to the second step under darkness which is an indication that they were not yet mature. It is necessary to mention that a successful maturation phase could produce differentiated organs on the same maturation medium in darkness. At such time it must be taken out to pursue their growth in the proliferation phase under illumination. As a fundamental principle, one meristematic cell or group of cells may be induced to develop single or multiple somatic embryos (George 1996). In date palm, typically there are two types of common somatic embryos: individual and multiple (Abul-Soad et al. 2004b). Both multiple somatic embryos and green shoots only were subjected to the proliferation phase and through the multiplication stage, whereas the individual somatic embryos were preferably left to grow by themselves. The latter type of somatic embryos grow rapidly to an intact shoot-root plantlet within 2–3 months in the rooting stage. Relying on such types of embryos can minimize the entire production-cycle time as compared to traditional shoot tip explants. In the case of rare male or female individuals, it is preferable to quickly regenerate plants rather than consume time in the multiplication stage of individual embryos to expedite mass production. 6.7 Direct and Indirect Formation of Organs by the Differentiation Process Friable callus is formed from initial cultured explants until the beginning of differentiation stage. Typically two structures were identified during the initial stage. Most prevalent were the globular structures which developed into green shoots or somatic embryos on the differentiation medium, as discussed above. These globular 102 A.A. Abul-Soad structures are like granules which are different in size; each is 1–3 mm in diameter and usually existing in clusters. Each cluster emerged from one floret. In the beginning, most of spikelet florets produced pro-embryonic masses (Fig. 6.2a) which are crystal white or often brownish yellow. These small masses multiplied and increased in number and became more organized. The growth and development was so quick as to make the initial explant of the spikelet appear as a swelled aggregate of these shiny globular structures (Fig. 6.2b). Also, the shiny brown color increased over time and the maturation of these globular structures. Therefore, the size of the globular structure clusters was gradual. The lower cluster on the spikelet explant was the largest, and then the size gradually decreased up to explant tip. It seems the endogenous hormonal content of each floret on the spikelet is different and due to this balance the nearest floret to the spikelet base produced more globular structures. Rarely all florets responded and produced these globular structures which indicate a nutritive competition among different florets on the spikelet explant leading to the death of un-responded florets. The second organized structure was direct shoot formation. Formation of direct shoots was rare in the inflorescence protocol. Moreover, produced shoots were unable to continue their growth and development to intact plantlets with roots. In addition, growth was slow compared to regular shoots which were produced from the globular structures after differentiation. However, it occurred when the initial explants were subjected to a long-incubation term in the state of physiological stress. Likewise this has been done with shoot-tip explants to induce direct organs by osmotic stress (Abul-Soad et al. 2007; Sidky et al. 2007). But the direct shoots of the shoot-tip protocol are so powerful and they can continue to growth vigorously. One of the advantages of the inflorescence technique is the normal character of the organized somatic embryos. Normal embryos (Fig. 6.3a) can be recognized by the white embryo coat which encloses a shoot and is connected with a thin white primary root. This coat splits longitudinally from the top producing a new green leaf. By continuing the embryo growth, the coat shrinks and turns brown. Sometimes, this coat produces embryogenic callus (Fig. 6.4). Mostly the normal embryos are twisted around themselves. There are two shapes for normal embryos: non-repeated embryos (Fig. 6.3b) and repeated embryos (Fig. 6.3c). Off-types of somatic embryogenesis are reported with shoot tip callus (Abul-Soad et al. 2004b). During the differentiation process of shoot-tip embryogenic callus, many somatic embryo shapes were differentiated. Determination of these shapes may be effective to improve the quality of produced plants and avoid abnormalities. Two shapes of somatic embryos were differentiated from the embryogenic callus (pro-embryos). One of them named repeated embryo (RE) and the other given the designation non-repeated embryo (NRE), or in other words, individual embryos. The RE is a cluster of embryos having the self-capability to proliferate additional secondary embryos and shoots. But the NRE directly differentiates into a single embryo which continues to grow into a distinct plantlet or can be used to form secondary embryos on its base or the periphery of the embryo coat (Fig. 6.3d). Growth depends on the nutrient medium composition. Using MS medium supplemented with 0.1 mg l–1 ABA and 0.5–1.0 mg l–1 BA produced secondary embryos on the NRE (Zaid 2003). 6 Micropropagation of Date Palm Using Inflorescence Explants 103 Fig. 6.3 Somatic embryos of inflorescence. (a) Normal and abnormal embryos, (b) Growth of an individual normal embryo. (c) A repeated embryo comprises more than 10 synchronized embryos. (d) Formation of secondary embryos on NRE having in their axils new colonies of embryogenic callus The origin of these secondary embryos could be the coat enclosing the embryo (Fig. 6.4a) or the base of an embryo at the connection area of enclosed shoot and primary root (Fig. 6.4b) or at any part of the embryo coat (Fig. 6.4c). It was observed that the outer white coat of a differentiated embryo splits vertically from the middle 104 A.A. Abul-Soad Fig. 6.4 Origin of secondary embryos. (a) On the coat split, (b) The connected area of enclosed embryo leaf and primary root, (c) Originates from the embryo coat. (d) Growth and development of secondary embryos on the base of embryo and white or creamy bright granules, which are in fact pro-embryos, were directly initiated on this split to form the secondary embryos. Secondary embryos develop from granules to somatic embryos with a white coat within 6–8 weeks (Fig. 6.4d). Mostly, abnormal embryos are smaller than normal (Fig. 6.3a). Some of these off-types were collected during the differentiation process and traced through their different stages of growth and development in vitro (Ibrahim 1999; Zaid 2003). 6 Micropropagation of Date Palm Using Inflorescence Explants 105 These off-types are designated to characterize their morphology: multi-tail, fused and multi-head (Zaid 2003). Therefore, using abscisic acid (ABA) in the maturation medium of date palm prevented maturing embryos from germinating precociously. The addition of 0.1–0.8 mg l–1ABA to the proliferation medium stimulated the process of secondary embryo formation, while increasing the concentration to 1.6 mg l–1 ABA was not effective (Hassan 2002). These off-types were not able to develop into complete plantlets and died (AbulSoad et al. 2004b; Zaid 2003). Using such off-types in the micropropagation led to embryogenic callus formation. Expectedly the embryos produced from such embryogenic callus can produce the somaclonal variation and originate off-types in the field. Thus, it is recommended that during the differentiation process, especially when shoot tip explants are used, these abnormalities be discarded to avoid any probability of variants formation (Abul-Soad et al. 2004b). Therefore, variety and subculture number during differentiation stage showed significant variance in abnormal production of shoot-tip embryogenesis. The percentage of abnormal shapes in somatic embryos was increased by augmenting the re-culture number. The Egyptian cvs. Sewi and Bent-Esha were higher than cv. Hiane in developing more abnormal shapes of somatic embryos. Moreover, the percentage of abnormalities increased from 64.78% in the first subculture of differentiation stage to 90.27% in the fourth subculture (Zaid 2003). Somatic embryos, which usually appear during the differentiation stage of the shoot-tip technique, were determined in a schematic illustration (Fig. 6.5). This classification is based on their morphology during the differentiation stage (AbulSoad et al. 2004b). It can be concluded that the micropropagation protocol utilized two subsequent subcultures (12 weeks) for the differentiation stage during which different shapes of somatic embryos were recognized. In the first subculture, the embryogenic callus (pro-embryos) which appearing as free crystal-white granules differentiated into predominant shapes: repeated embryos and non-repeated embryos. The NRE was an individual embryo 5–10 mm in length and fast in growth and development to green leaves (Fig. 6.3b). On the other hand, the RE is a granule developed in a cluster of embryos, all of them gathered at the same point of origin. These embryos often number 10–15 and are synchronized, i.e. having different sizes indicating different ages (Fig. 6.3c). These two shapes are typically similar to the differentiated somatic embryos of the inflorescence technique. In the second subculture, the RE and NRE were transferred to the same nutrient medium to trace their growth and development. It is worth mentioning that picking up these germinating embryos in the first subculture stimulated further germination of remaining granules into new somatic embryos as well as new granules. This may indicate the synchronization in the differentiation process, i.e. earlier differentiated embryos prohibited the development of other matured pro-embryos to proceed to grow and become intact somatic embryos. The second subculture allowed fast growth of individual embryos to whole plantlets (5–10 cm in length) after 6 weeks in culture, which were transferred directly to the pre-rooting stage (Abul-Soad et al. 1999). 106 A.A. Abul-Soad White embryogenic callus (granules) Sub-culture 1 Differentiation medium resulted in two types of embryos Repeated embryos (RE) Sub-culture 2 Culturing on the same differentiation medium (Regenerative types) A cluster of embryos Non-repeated embryos (NRE) (Two types of the individual embryos) A cluster of shoots Proliferated embryo (PE) Non-proliferated (NPE) Excised embryos which have primary root Excised embryos which have primary root To multiplication stage To pre-rooting stage Fig. 6.5 Schematic illustration of shoot-tip embryogenic callus differentiation during two subcultures. The differentiation medium was composed of MS-basal medium supplemented with NAA 0.1 mg l–1, 2iP 0.1 mg l–1 and activated charcoal 100 mg l–1 (Abul-Soad et al. 2004b) This type of NPEs were healthy and rapidly growing to intact plantlets compared to the plantlets which will be separated from multiplying clusters at the end of multiplication stage. Sometimes, an embryogenic callus produced on the basal part of this individual embryo (NRE), is called a proliferated embryo (PE) (Fig. 6.4b). The PE of the second subculture was used in the multiplication stage with the RE of the first subculture as multiplying types in the multiplication stage (Fig. 6.3c). Classification of somatic embryos into these categories during the differentiation stage and directing them into the multiplication stage or the rooting stage is crucial for commercial production of in vitro date palms. No morphological difference was observed between the differentiated embryos of shoot tip or inflorescence explants. Moreover, abnormal embryos were rarely discovered during the differentiation process of the inflorescence culture (Fig. 6.3a). According to the inflorescence technique used, the overwhelming majority of emerged somatic embryos were normal (Fig. 6.3a). The abnormal somatic embryos must be discarded to avoid somaclonal variations. The intervening callus phase in the shoot-tip technique could be the reason for abnormalities as compared to the current inflorescence technique without an unfriable callus phase. More details on 6 Micropropagation of Date Palm Using Inflorescence Explants 107 types of somatic embryos and abnormality in date palm are provided by Abul-Soad et al. (2004b). Differentiation of the new organs, i.e. green shoots (Fig. 6.2d) and somatic embryos are associated with direct emergence of new embryogenic callus. Typically the color of this embryogenic callus is white or sometimes creamy. Its organogenic potential is high by continuous subculture onto the same differentiation medium resulted in new somatic embryos as well as production of embryogenic callus without loss of the organogenic potential. This process occurred through 6–7 subcultures each from 3–4 weeks in semi-log plot, i.e. the amount of friable callus was low in the beginning of the differentiation stage, then increased to the highest level by increasing the subculture up to number 7, then decreased and eventually no additional friable callus formed, and the differentiation stage was ended at subculture 13 (Abul-Soad and Mahdi 2010). In embryogenic callus derived from shoot-tip explants, the events occur just the opposite at the differentiation stage. The newly differentiated callus rapidly loses organogenic potential after only 4–6 subcultures. This could be due to habituation of callus cells on the same composition of the nutrient medium. Eventually after 4–6 re-cultures of the shoot-tip embryogenic callus and side process of new callus formation, a big amount of unfunctional friable callus was achieved. The new clones of callus were morphologically similar to the ordinary embryogenic callus, white granules and friable but lost the ability to development to intact somatic embryos. Sometimes this type of callus produces tiny and abnormal embryos. 6.8 Proliferation During the Multiplication Stage As mentioned earlier, multiple embryos and green shoots after differentiation would were transferred onto the proliferation medium at the multiplication stage (Fig. 6.6a). During the proliferation process no secondary unfriable callus was observed. This callus is globular and variable in size (1–2 mm), always initiating in leaf axils as a source of new shoots. It is total caulogenesis (shoot forming) and sometimes with a minimally intervening embryogenic callus phase (the same shining globular structures). Simultaneously proliferation via caulogenesis was associated with secondary somatic embryo formation. The origin of this was the regular shining globular structure which formed in the axils of juvenile leaves. The proliferated somatic embryos easily separate from the multiplied shoot cluster (Fig. 6.6a). Then these individual somatic embryos will be transferred directly to the rooting stage. According to the knowledge of the author, no research has been performed to determine the maximum number of subcultures in the multiplication stage. However, it is recommended that a lower number of subcultures will always be better. In fact, the maximum number of subcultures in the multiplication stage is not definite. However, as a specific example, the cultures of inflorescence were multiplying well throughout 13 subcultures done at the Date Palm Research Institute, Khairpur, Pakistan, during the period 2008–2010. Also, there were produced from one inflorescence of cv. Gulistan; it provided 10,000 healthy plantlets in the rooting stage and partially transplanted 108 A.A. Abul-Soad Fig. 6.6 Multiplication, rooting and acclimatization stages. (a) Proliferation of multiple embryos and individual embryos, (b) Plantlets during rooting stage, (c) Direct shoot formation on spikelet initial explant after 11 subcultures, (d) 3, 6, 12 and 18 month stages of growing plants into the greenhouse. It is very important to mention that for over one century the population of recalcitrant cv. Gulistan at the Dera Ismail Khan District, stood at 1,000–2,000 trees due to the limitation of new offshoots through traditional propagation. Notwithstanding, within only 2 years it became possible to produce that number and more from a single inflorescence. This is strong justification for using the promising inflorescence technique. 6 Micropropagation of Date Palm Using Inflorescence Explants 6.9 109 Formation of Healthy Plantlets In this protocol, three steps were taken to produce a good root-shoot system (Fig. 6.6b), whereas the fourth step is to pre-acclimatize (in vitro hardening protocol) the plantlets in vitro to reduce ex vitro stress in the greenhouse. •฀ Well-rooted plantlets are achieved on a medium comprised of ¾ MS salt strength and 50 g l–1- sucrose (Abul-Soad et al. 1999; Ibrahim et al. 1999b). This nutrient medium supplemented with 0.1 mg l–1 NAA to improve the root system and 0.05 mg l–1 BA to enhance leaf formation (Omar 1988a). Also, 1.0–2.0 mg l–1 Ca-panthothianate was added in order to prevent necrosis and vitrification. The base of culture tubes may be covered with aluminum foil to reduce light emission by which growth and development of root system can be improved. The base covering can be done for the entire rack of tubes (Abul-Soad et al. 1999). •฀ Plantlets are transferred from standard small tubes (25 × 150 mm diameter and height, respectively) to larger tubes (28 × 250 mm) to allow erect shoot growth. Plantlets should be maintained on this medium until they reach a suitable height with a good root system and 2–3 leaves. Typically this occurred at 3 months (2 re-cultures) at least on such rooting medium. About 80–90% of cultured plantlets on this medium produced a fair root system (Abul-Soad et al. 1999). Whereas, it is reported that 30–40% of plantlets cultured on agar media, containing only 0.1 mg l–1 NAA only produced prolific adventitious root growth (Tisserat 1982). •฀ In the third step, better growth responses of date palm plantlets are associated with the addition of 3 g l–1 AC to the medium (Abul-Soad et al. 2006). Generally, low rates of photosynthesis in plantlets grown in vitro have been attributed to low light conditions. Thus, increasing the light level to 9,000 lm m–2 strongly enhanced growth (Ibrahim et al. 1999a). 6.10 In Vitro Hardening Protocol The process of acclimatization can begin while plantlets are still in vitro. In vitrogrown plantlets grow under the controlled growing conditions such as low light levels, aseptic conditions, medium containing sugar and nutrients to allow for heterotrophic growth and in high humidity. They are unable to survive when transferred directly to the greenhouse where low humidity levels and opened stomata of newly-transplanted plantlets allow intensive transpiration which leads to rapid drying of leaves. Ex vitro date palm plants which were newly shifted to the greenhouse without keeping the humidity above 90% could not resist the humidity deficiency more than half an hour. After that, leaves shrink and became like thin thread. Therefore, leaves could not be able to return back to their natural state after shifting them to high humidity conditions (85–95%). Thus it is advisable to pre-acclimatize the plantlets gradually while they are in vitro (Abul-Soad et al. 1999). 110 A.A. Abul-Soad •฀ In this step, plantlets are rinsed with sterile distilled water in order to remove excess adherent media under aseptic conditions, and then cultured on a medium free of sugar, half strength of MS and in culture tubes capped with aluminum foil. Cultures are kept under a high light intensity (9,000 lm m–2). •฀ In this procedure, ventilation between the inside and outside of the tube was increased through gradually punching holes in the aluminum foil, followed by complete removal a few days before transplanting. This gradual approach is beneficial to reduce the relative humidity in the tubes and to increase epicuticular wax development on the leaves. At the same time, omission of sucrose from the nutrient medium brings about a shift from heterotrophy to autotrophy (Abul-Soad et al. 1999). Hereafter, high rates of photosynthesis along with a high light level and adequate gas exchange become paramount for survival in the greenhouse. The same in vitro hardening procedure has been used in other crops (Kozai et al. 1987). All of these factors help to acclimatize date palm plantlets gradually to ensure survival in the greenhouse. Date palm plants are given the chance to rely on themselves to respond like natural seedlings in the field and produce all their requirements from carbohydrates without any addition of chemical elements during the first 1–2 months in the greenhouse. Another method of in vitro hardening in date palm has been described wherein when plantlets reach a desired size (15 cm in length), they are hardened by subculturing into minimal organic medium (MS salts; inositiol, 100 mg l–1; thiamine-HCl, 0.4 mg l–1; sucrose, 30 g l–1) for 2 weeks with incubation at 10,000 lm m–2 illumination (Omar 1988b). It was observed that the typical twisted shoots became erect, and the pale green color became dark green. Moreover, in the case of strong plantlets which have 4–5 adventitious roots and wide, erect and dark green leaves 15–20 cm in length, the closure may be pushed up by plantlet tip. At the end of the pre-acclimatization stage, the wilting of the leaves when they are in uncovered tubes represents a positive sign for early in vitro detection of inadequate plants. Such plantlets must be discarded to avoid further effort in transplanting and acclimatization in the greenhouse. Plantlets with mostly erect leaves were shifted to soil beds under a low transparent plastic tunnel to keep the relative humidity at 80–90%. The reason for the change is to reduce fungal infection as compared to the more common range of 90–100%. 6.11 Transplanting Specialized environmental conditions, high humidity and low photosynthesis of in vitro culture can result in the formation of plantlets exhibiting abnormal morphology, anatomy and physiology. After ex vitro transfer, plantlets might easily be impaired as well by sudden changes in environmental conditions and therefore a period of acclimatization is needed to overcome some abnormalities (Pospisilova et al. 1999). 6 Micropropagation of Date Palm Using Inflorescence Explants 111 Healthy shoot-root system plantlets of date palm are transplanted on different soil beds in the greenhouse for acclimatization. Many soil mixtures have been used as a substrate for date palm acclimatization (Abul-Soad et al. 1999; Al-Jibouri et al. 1988; Hegazy et al. 2006; Madhuri and Shankar 1998). The highest plantlet survival rate after 3 months was 93%, planted under the current procedure of transplanting (Fig. 6.6d). Plantlets selected for this procedure should have 2–3 erect leaves of an average 10–20 cm height with well-developed adventitious root systems. These plantlets then were transplanted into special pots (5 cm diameter × 18 cm height) containing vermiculite as the soil bed. Placement of small stones at the bottom of pots promoted root growth due to effective drainage of irrigation water. 6.12 6.12.1 Acclimatization Procedures •฀ Ideal plantlets within long tubes are transferred from the laboratory to a greenhouse and left for at least 1–2 h under the greenhouse environment which has a higher temperature of around 30oC, relative humidity of 40–60% and natural illumination (daylight without direct sun) 10,000–25,000 lm m–2. The nutrient media stuck around the root system was washed off by immersion in sterilized water and carefully shaken. •฀ Plantlets are dipped in 3–5 g l–1 systematic fungicide (Topsin M 70) solution for 1–2 min. •฀ Direct planting in suitable pots (5 × 18 cm) filled with autoclaved vermiculite and making note on the container of the associated data of the cultivar name. •฀ Planted pots placed under low transparent plastic tunnels one by one within a few min to avoid humidity decrease inside the tunnel associated with spontaneous opening of the tunnel to put in the plants. It is preferable to construct a tunnel to contain 250 plants as a maximum number which can be handled within 1–2 h. Growing conditions within the tunnel are maintained at 30–35oC and 90–100% relative humidity. These conditions are gradually reduced by placing the plants within the tunnel in parallel with the ventilation process until the plants are fully exposed to the natural growing conditions within the open greenhouse after 1–2 months when the plastic sheet is removed. •฀ The tunnel is kept closed after placement of the pots and left undisturbed for 3–7 days. During this period irrigation is prohibited. •฀ The tunnel is opened after 7 days for 10–15 min of ventilation and a Copper OxyChloride spray treatment of 3 g l–1 solution fungicide applied. Also, dead plants are removed and leaves infected with fungus are cut off. •฀ Gradual opening of the tunnel after 2 weeks of planting for the subsequent 2 weeks. 112 A.A. Abul-Soad •฀ Fertilization and irrigation process after 2–3 months of planting as required (Fig. 6.6d). Until now no work has been done to determine the actual requirements of these growing date palm plants from different elements. However, 2–3 g l–1 N-P-K fertilizer (17-17-17) gave good results. Also, using the mycorrhiza (Bouamri et al. 2006) and humic acid which is a natural chelating soil mobilizing agent was effective. •฀ Transfer successful plants into larger pots 20 × 25 cm. Fertilizer dose increased slightly. Growing plants must be fertilized with irrigation water containing gradual doses of a chemical fertilizer to encourage the plant growth. The program of fertilization beginning after 2–3 months of transplanting with light dose 2–3 g l–1 N-P-K fertilizer (17-17-17, Zarkhaze) every 1–2 weeks. Every 1–2 months an additional application of Copper OxyChloride 3 g l–1 over 6–18 months (Fig. 6.6d). Plants 50 cm in height are then transferred to the nursery or directly planted in the field. Plants at 12–18 months old begin to produce feathered (compound) leaves. When plants push thick white roots through the pores at the pot bottom after 5–6 months, they should be transferred to a larger pot. Growing plants can be placed on a wooden base or plastic sheet to prevent root extension into underlying soil. 6.12.2 Most Important Factors in Date Palm Acclimatization •฀ Pre-acclimatization on a low-nutrient liquid medium while plantlets are kept under laboratory conditions. •฀ Use of a sterilized soil bed and fungicide Topsin M 70 application before transplanting in the greenhouse. •฀ Maintain 85–90% relative humidity and 30–35ºC temperature in ambient environment during the first 7–10 days of transplanting to avoid leaf wilting and fungus infection. 6.12.3 Expected Problems and Their Solutions •฀ Quick wilting of leaves while transplanting. Rapid planting process within the shortest time 1–2 min and pre-acclimatizes the plants while they are in the lab. •฀ Fungal infection of basal part of the plant and leaves separation while their root may be still healthy, white and inside the soil. Always wash the plants thoroughly with sterilized water before planting. Also, keep the basal part of the plant completely above the pre-sterilized soil surface. Treatment with a systematic fungicide (Topsin M 70 or any other fungicide) solution is beneficial after 1 week of transplanting. •฀ Fungal infection on leaves, mostly during the first 2 weeks of acclimatization. It is caused by condensation of water drops falling inside the tunnel. Tunnel should not be opened during the first 3–7 days. In addition, dipping the plants in a systematic 6 Micropropagation of Date Palm Using Inflorescence Explants 113 fungicide solution for 1–2 min before planting, without washing after treatment. Also, spraying with Copper OxyChloride solution is helpful after 1 week of transplanting. •฀ Dead and infected outer leaves during first month. Cut them off then spray with Copper OxyChloride solution 5 g l–1. •฀ Low humidity in the tunnel. Keep the temperature in the range of 30–35ºC. •฀ Leaves with late fungal infection. Relative humidity should be gradually reduced after a maximum 20 days of transplanting and kept at 50–60%. 6.13 Fruit Type in Open-Field There are some reports from Tunisia and Morocco referring to fruiting of date palms derived from the inflorescence. INRA-J19 a selected date palm genotype which was field planted in Marrakech (Morocco) began to produce fruits in 2005. This is providing information on true-to typeness and consequently the applicability of such a technique for mass propagation of date palm (Abahmane 2007). The Plant Tissue Culture Laboratory, Al-Ain City, UAE announced in 2005 the early emergence of male spathes of an extraordinary male date palm micropropagated from an inflorescence. Furthermore, the average number of spathes per plant was about 2–3, while the percentage of spathe production was 96%, i.e. 67 male plants produced spathes out of the original 70 plants (El-Korchi 2007). However, more investigations are needed to ensure the genetic stability of inflorescence in vitro-derived plantlets. Also, evaluation of fruit quality in the field should be carried out similar to work performed with shoot tip in vitro-derived plantlets (Abd-Allah et al. 2008). 6.14 Conclusion and Prospective Research work has been ongoing for a few decades to use inflorescence explants in micropropagation of date palm. However, commercial laboratories are using shoot tip explants despite the competitive benefits of inflorescence buds. This chapter explores these benefits and gives a detailed procedure for how to use inflorescences in the micropropagation of date palm. Immature inflorescence culture involves sterilization of small spikes and culturing segments on nutrient media according to the culture stage described in Table 6.1. Direct formation of globular structures occurs, pro-embryos or direct shoots formation (Fig. 6.6c). A histology study is required to further clarify the developmental process involved. Resultant somatic embryos can be classified into two categories, individual and multiple somatic embryos. Directing these embryos to the multiplication or rooting stage is obligatory. The multiplying cultures are used in the multiplication stage to get the maximum number of shoots which are then directed to the rooting stage. Rooted plantlets are subjected to an 114 A.A. Abul-Soad in vitro hardening procedure before transplanting in the greenhouse. The question of when to stop the cycle of in vitro multiplication to produce the maximum number of high quality true-to-type plantlets remains unanswered. Studying the genetic stability of in vitro plantlets could enhance consumer confidence in relation to commercial production through avoiding somaclonal variation which may result of in prolonged cultures. Also, it would be of importance in studying genetic stability in relation to various culture stages. 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